Abstract
Retinaldehyde dehydrogenase II (RalDH2) converts retinal to the transcriptional regulator retinoic acid in the developing embryo. The x-ray structure of the enzyme revealed an important structural difference between this protein and other aldehyde dehydrogenases of the same enzyme superfamily; a 20-amino acid span in the substrate access channel in retinaldehyde dehydrogenase II is disordered, whereas in other aldehyde dehydrogenases this region forms a well defined wall of the substrate access channel. We asked whether this disordered loop might order during the course of catalysis and provide a means for an enzyme that requires a large substrate access channel to restrict access to the catalytic machinery by smaller compounds that might potentially enter the active site and be metabolized. Our experiments, a combination of kinetic, spectroscopic, and crystallographic techniques, suggest that a disorder to order transition is linked to catalytic activity.
Highlights
In the developing embryo the generation of retinoic acid by metabolism of the inactive precursor retinol must be finely regulated
Amino acids ϳ457– 477 were not visible in the electron density maps [13] and unlike their counterparts in the previous ALDH structures, which were determined in the presence of co-factor but absence of substrate (9 –12, 15) were disordered. (Alternatively, these regions may have more than one possible conformation, and these conformations could be randomly distributed in the tetramer of the asymmetric unit.) In contrast to what is observed for ALDH2 and ALDH3, retinaldehyde dehydrogenase type II shows preference for aldehydes with long hydrocarbon chains in vitro and does not efficiently oxidize acetaldehyde [8]
To test this theory we chose to introduce two mutations in the mobile loop: residues Leu-458 and Asn-459 were replaced by a Phe and Gly, respectively, their counterparts in the highly homologous bovine mitochondrial dehydrogenase [10]. Studies with this enzyme by others have provided an enormous amount of mechanistic information that we can utilize to guide our interpretation of data obtained for RalDH2 (10, 19 –26)
Summary
Bacterial Strains and Media—The expression strain E. coli BL21(DE3) pLysS from Novagen was used to overexpress all RalDH2 constructs. To 128 mM for acetaldehyde) in an Applied Photophysics stopped-flow reaction analyzer by monitoring NADH fluorescence (excitation wavelength 340 nm, emission cutoff filter 420 nm) with a 0.2-cm path length These experiments were performed at 25 °C. Measurements were performed at either 230 or 460 nM enzyme in the presence of 1 to 4 mM NAD, 10 mM HEPES, pH 8.5, 150 mM KCl, 1 mM EDTA, and 2 mM -mercaptoethanol These concentrations are those in the observation cell. Because this enzyme is incapable of producing NADH, the lack of a signal is consistent with our interpretation that the fluorescence observed is that of NADH, which results from either resonance energy transfer from Trp or from NADH absorbance at 295 nm These experiments were performed with 460 nM enzyme, 4 mM NAD, 250 M octanal in the observation cell
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